The synthesis of carbamyl phosphate, an intermediate in pyrimidine and arginine biosynthesis, is catalyzed by ammonia and glutamine dependent carbamyl phosphate synthetase (CPS). E coli has a single glutamine dependent CPS composed of a glutaminase (alpha subunit) and synthetase (Beta subunit). The two subunits are encoded by the carAB operon. The alpha subunit is a member of a large family of enzymes that provide amide nitrogen for diverse biosynthetic reactions. The Beta subunit probably evolved by duplication of the same ancestral gene that gave rise to carbamate synthetase. This enzyme has a complex mosaic structure with active sites related to the biotin containing carboxylases. Studies of carAB and site-directed mutants have enabled us to map the functional, regulatory, and subunit contact domains in the enzyme. The enzymatic and substrate binding properties of structurally native but catalytically impaired CPSs have allowed separate sequential steps of the reaction mechanism to be ascribed to two-nucleotide binding domains located separately in each duplicated half of the Beta subunit. The catalytic site mutants have also revealed reciprocal modulatory influences of active site domains located in the alpha and Beta subunits and have helped to identify some key residues underlying these interactions. These and earlier biochemical data of E coli CPS have contributed a good working model for more detailed probings of the structure-function relationships in the enzyme. Four objectives will be pursued during the coming period. Previous attempts to crystallize CPS have been sufficiently encouraging to warrant more intensified efforts along these lines. A major part of our time will be devoted to obtaining stable and highly diffracting crystals of CPS and/or of its constituent subunits. A collaborative arrangement has been established with Dr. Wayne Hendrickson's laboratory to solve the structure. We will also continue our studies of carB mutations affecting the partial reactions and active site coupling properties of the Beta subunit. These studies are anticipated to provide additional mechanistic details and identify catalytically important residues. The proposal that the catalytic mechanism of type G glutaminases is similar to that of cysteine proteinases has gained support from the demonstration of a thioester intermediate during the hydrolysis of glutamine and the observed sequence homology of the alpha subunit to papain. This model will be experimentally tested through studies of site-directed mutations in residues conserved among the two groups of proteins, particularly those residues known to affect the stability of cys-his ion pairing in papain. Finally, experiments are proposed to examine IMP and UMP induced quaternary conformational changes by the carboxyl proximal allosteric domain and of the resultant contribution by this domain of residues necessary for substrate and metal binding at the active sites of the Beta subunit.